U.S. patent number 11,215,623 [Application Number 15/671,663] was granted by the patent office on 2022-01-04 for assays for detecting the presence or amount of an anti-drug antibody.
This patent grant is currently assigned to GENZYME CORPORATION. The grantee listed for this patent is Genzyme Corporation. Invention is credited to Ryan Grabert, Susan Richards, Valerie Theobald, Yuanxin Xu, Jad Zoghbi.
United States Patent |
11,215,623 |
Grabert , et al. |
January 4, 2022 |
Assays for detecting the presence or amount of an anti-drug
antibody
Abstract
Methods and kits for detecting antibodies (e.g., anti-drug
antibodies). Such methods and kits permit the detection of, for
example, anti-drug antibodies in human body fluids, such as blood,
plasma and serum.
Inventors: |
Grabert; Ryan (Bridgewater,
NJ), Richards; Susan (Bridgewater, NJ), Theobald;
Valerie (Bridgewater, NJ), Xu; Yuanxin (Bridgewater,
NJ), Zoghbi; Jad (Bridgewater, NJ) |
Applicant: |
Name |
City |
State |
Country |
Type |
Genzyme Corporation |
Cambridge |
MA |
US |
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Assignee: |
GENZYME CORPORATION (Cambridge,
MA)
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Family
ID: |
52684652 |
Appl.
No.: |
15/671,663 |
Filed: |
August 8, 2017 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20180088140 A1 |
Mar 29, 2018 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14619422 |
Feb 11, 2015 |
9759732 |
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61938556 |
Feb 11, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
33/539 (20130101); G01N 33/6854 (20130101); G01N
33/5306 (20130101); G01N 33/94 (20130101); G01N
2430/00 (20130101) |
Current International
Class: |
G01N
33/53 (20060101); G01N 33/539 (20060101); G01N
33/94 (20060101); G01N 33/68 (20060101) |
References Cited
[Referenced By]
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Apr 2010 |
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JP |
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1990/006515 |
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Jun 1990 |
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WO |
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2007/101661 |
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Sep 2007 |
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WO |
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WO |
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Jul 2009 |
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WO |
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WO 2012154253 |
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WO |
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2013/132000 |
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Sep 2013 |
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WO |
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WO 2015123315 |
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Aug 2015 |
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WO |
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Other References
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|
Primary Examiner: Counts; Gary
Attorney, Agent or Firm: Lathrop GPM LLP Velema; James H.
Stone-Hulslander; Judith L.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser.
No. 14/619,422, filed Feb. 11, 2015, now U.S. Pat. No. 9,759,732,
which claims the benefit of U.S. Provisional Patent Application
Ser. No. 61/938,556, filed Feb. 11, 2014, the entire contents of
which are hereby incorporated by reference.
Claims
What is claimed is:
1. A method for determining the presence or absence of an anti-drug
antibody (ADA) in a sample, the method comprising: contacting the
sample with an excess amount of drug to which the ADA binds to
saturate the ADA and to form drug/ADA complexes; contacting the
drug/ADA complexes with polyethylene glycol (PEG), to form a
precipitate comprising drug/ADA complexes; contacting the
precipitate with a basic solution to dissociate the drug/ADA
complexes, thereby forming dissociated ADAs and dissociated drugs,
wherein the basic solution causes the solution of dissociated ADAs
and dissociated drugs to have a specific basic pH; immobilizing the
dissociated ADAs on a substrate under conditions where dissociation
of the ADAs and the drugs is maintained, wherein the conditions
where the dissociation of the ADAs and the drugs are maintained is
that the solution of dissociated ADAs and dissociated drugs is
maintained at the specific basic pH; and determining if the ADA is
present or absent in the sample.
2. The method of claim 1, wherein the determining step comprises
contacting the immobilized ADA with drug labeled with a detectable
label; and determining the presence or absence of said detectable
label, to thereby determine the presence or absence of ADA in the
sample.
3. The method of claim 1, wherein the substrate comprises a porous
carbon surface.
4. The method of claim 1, wherein the substrate comprises a carbon
surface, glass surface, silica surface, metal surface, a polymeric
material, a surface containing a metallic or chemical coating, a
membrane, a bead, a porous polymer matrix, or substrates comprising
cellulosic fibers, or any combination thereof.
5. The method of claim 2, wherein the detectable label comprises a
label selected from the group consisting of a radioactive isotope,
an enzyme, a fluorescent label, a chemiluminescent label, an
electrochemiluminescent label, and a substrate for an enzymatic
detection reaction.
6. The method of claim 1, wherein the drug comprises an antibody or
functional fragment thereof, nucleic acid, peptide, polypeptide,
peptidomimetic, carbohydrate, lipid, an organic small molecule
compound, an inorganic small molecule compound, or any combination
thereof.
7. The method of claim 1, wherein the drug is a drug modified to
exhibit less immunogenicity as compared to the same drug in
unmodified form.
8. The method of claim 1, wherein the PEG comprises at least one
PEG selected from the group consisting of PEG1000, PEG1450,
PEG3000, PEG6000, PEG8000, PEG10000, PEG14000, PEG15000, PEG20000,
PEG250000, PEG30000, PEG35000, and PEG40000.
9. The method of claim 1, wherein the sample is contacted with PEG
at a concentration of between about 0.1% and about 10.0%.
10. The method of claim 1, wherein the sample comprises the
drug.
11. The method of claim 1, wherein the method further comprises
immobilizing the drug on the substrate before or after the step of
immobilizing the ADA on the substrate.
12. The method of claim 1, wherein the sample comprises a
biological sample, wherein the biological sample comprises material
selected from the group consisting of body fluids, mucus
secretions, saliva, blood, whole blood, plasma, and serum.
13. The method of claim 1, wherein the basic solution comprises an
organic base, an inorganic base, or a mixture thereof.
14. The method of claim 1, wherein the basic solution comprises a
base at a concentration of between about 0.1 M to about 5 M.
15. The method of claim 1, wherein the basic solution comprises a
base selected from the group consisting of urea, sodium hydroxide,
rubidium hydroxide, cesium hydroxide, calcium hydroxide, strontium
hydroxide, barium hydroxide, zinc hydroxide, lithium hydroxide,
acetone, methylamine, ammonia, and any combination thereof.
Description
TECHNICAL FIELD
This invention relates to methods and kits for detecting the
presence of anti-drug antibodies in a sample, and more particularly
to methods and kits for detecting anti-drug antibodies in the
presence of a drug in the sample.
BACKGROUND
The introduction of biotherapeutics (e.g., biologic agents such as
proteins, peptides, nucleotides, etc.) has given a major boost to
the treatment of diseases such as inflammatory bowel disease,
ankylosing spondylitis, multiple sclerosis and rheumatoid
arthritis. In many cases these biological agents have proven very
successful in clinical practice. Biologic agents, including
therapeutic antibodies, are known to have immunogenic potential,
and administration of therapeutic proteins to a patient can induce
immune response leading to the formation of anti-drug antibodies
("ADAs"). Such ADAs may reduce the effectiveness of the therapeutic
protein. For example, they may bind to or/and neutralize the
therapeutic protein, resulting in changes of drug pharmacokinetics
or pharmacodynamics that alters drug efficacy. ADAs may cause
serious side effects, including allergic reactions,
cross-reactivity against endogenous proteins by neutralizing
antibodies (NAbs), and complement activation. The production of
ADAs have been described for several monoclonal antibodies
available for the treatment of rheumatoid arthritis (adalimumab and
infliximab), Crohn's disease (infliximab), multiple sclerosis
(natalizumab and alemtuzumab) and plaque psoriasis (adalimumab). In
some patients, the clinical benefits provided by such therapeutic
proteins diminishes over time due to the formation of ADAs.
Immungenicity risk assessment is critical to understand frequency
and severity for drug induced ADA. NAb cross-reactive to endogenous
protein causing depletion syndrome has been reported
(erythropoietin).
With an increasing number of therapeutic proteins approved for
clinical use, the immunogenicity of such products has become
informative to clinicians, manufacturers and regulatory agencies.
It is well-established that certain substances will affect the
detection or quantitation of an analyte in immunoassays (or ligand
binding assays). These interference factors including but not
limited to circulating drugs negatively impact assay specificity,
accuracy, and sensitivity. "Drug interference" that reduce ADA
assay "drug tolerance" is regarded as a major technical challenge
for immunogenicity assessment to monitor ADA as part of patient's
monitoring for drug clinical safety and efficacy.
Although the above approaches demonstrated some improvement in drug
tolerance, sensitivity and relative accuracy is not maintained in
comparison to no-drug ADA detection therefore risking false
negative and under-reporting ADA incidence and titers in treated
patients. Despite industry regulatory guidance documents and white
papers recommending sensitivity between 250 and 500 ng/mL [Shankar
G, Devanarayan V, Amaravadi L et al.: Recommendations for the
validation of immunoassays used for detection of host antibodies
against biotechnology products. Journal of Pharmaceutical and
Biomedical Analysis 48(5), 1267-1281 (2008); Mire-Sluis A R,
Barrett Y C, Devanarayan V et al.: Recommendations for the design
and optimization of immunoassays used in the detection of host
antibodies against biotechnology products. Journal of Immunological
Methods 289(1-2), 1-16 (2004)], drug tolerance is sometimes
evaluated without any acceptance criteria and clinical protocols
are then written instructing long wash-out periods before antibody
measurement to allow for drug clearance and the avoidance of false
negative results due to drug interference. This approach is not
desired due to risks in missing ADA assessment in early time points
especially in the case with a long half-life drug and/or
multi-dosing regimen and the wash out period approach is not
feasible. Some non-ligand binding based methods such as mass
spectrometry has been evaluated for PK in the presence of ADA
interference, the expected assay sensitivity has not been
acceptable and enrichment of analyte is needed which is ligand
binding based which poses the similar challenges.
A variety of assay formats have been used with success to detect
ADAs, including ELISA (direct, indirect and bridging),
radioimmunoassays, electrochemiluminescence, and surface plasmon
resonance. The development of such assays, however, is often
complicated by interference caused by the presence of the drug. The
challenge of analytical interferences in ligand binding assays has
long been recognized. With the advent of long-lived monoclonal
antibody therapies, the need for specific techniques to detect ADA
in the presence of drug is of particular concern. The most widely
adopted approaches in use currently still have limitations of
timing, sensitivity or accuracy. Thus, there is a need in the art
for methods and kits to more accurately and reproducibly detect the
presence of ADA in samples, such as biological samples.
SUMMARY
The present invention is based, at least in part, on the discovery
of a novel assay method that is effective for reducing or
eliminating the problems caused by interference by drug or target
in ADA detection. In particular, the present invention is based on
the development of a novel ADA assay comprising the following
exemplary steps. First, excess drug material is added to the
samples containing potential ADAs (both free ADA and ADA/drug
complex) to bind all remaining free ADAs, forming drug/ADA
complexes. Second, these complexes are precipitated using
polyethylene glycol. Third, after a series of washes to remove
serum protein and immuoglobulin, the final precipitate (drug/ADA
complexes) is reconstituted with a solution to dissociate the
complexes and then coated on a large surface (under conditions to
keep drug and ADA apart) or substrate (e.g., a high bind carbon
plate with high coating capacity) for a time sufficient to allow
coating of all dissociated free drugs and free ADAs. Fourth,
specific detection of the total ADA levels is then performed using
labeled drug. Accordingly, the present invention relates to
methods, compositions and kits for determining the presence or
amount of an ADA in a sample (e.g., a biologic sample).
In one embodiment, the final precipitate (drug/ADA complexes) is
reconstituted with an acid solution to dissociate the complexes and
then coated on a large surface (under acidic conditions to keep
drug and ADA apart) or substrate (e.g., a high bind carbon plate
with high coating capacity) for a time sufficient to allow coating
of all dissociated free drugs and free ADAs. The acidic environment
prevents the complexes from reforming while being immobilized onto
the substrate surface. When an acid solution is used to dissociate
the complexes, the assay can be referred to as a PandA (PEG and
Acid) assay.
In another embodiment, the final precipitate (drug/ADA complexes)
is reconstituted with a basic solution to dissociate the complexes
and then coated on a large surface (under basic conditions to keep
drug and ADA apart) or substrate (e.g., a high bind carbon plate
with high coating capacity) for a time sufficient to allow coating
of all dissociated free drugs and free ADAs. The basic environment
prevents the complexes from reforming while being immobilized onto
the substrate surface.
The selection of an acidic or basic solution will depend on the
parameters of the drug (e.g., the biologic drug), such as pI, or
the presence of certain conjugating bonds, and the selection will
have minimal effect on the integrity and structure of the drug.
In some embodiments, the incubation step following the initial acid
or base addition can be carried out at 22.degree. C., 23.degree.
C., 25.degree. C., 27.degree. C., 30.degree. C., 32.degree. C.,
35.degree. C., 37.degree. C., 39.degree. C. or higher.
In some embodiments, following the final precipitation step, each
sample can be further diluted to a final sample dilution of, e.g.,
1:20, 1:25, 1:30, 1:40, 1:50, or 1:60.
In one aspect, the disclosure provides a method for determining the
presence or absence of an ADA in a sample, the method comprising
contacting the sample with an excess amount of drug to which the
ADA binds to form drug/ADA complexes, contacting the drug/ADA
complexes with polyethylene glycol (PEG), to form a precipitate
comprising drug/ADA complexes, contacting the precipitate with a
solution to dissociating the drug/ADA complexes, immobilizing the
dissociated ADAs on a surface and/or substrate, and determining the
presence of or amount of said ADA. In a further aspect, the
determining step comprises contacting the immobilized ADA with drug
conjugated with a detectable label, and determining the presence of
or amount of said detectable label, to thereby determine the
presence or amount (titer) of ADA in the sample.
In some embodiments, the method for determining the presence or
absence of an ADA in a sample further comprises before, after or
part of the determining step, determining the amount of ADA in the
sample.
In some embodiments, the method for determining the presence or
absence of an ADA in a biological sample further comprises, after
the immobilizing step, treating (e.g., washing) the support to
remove unbound drug.
In other embodiments, the method for determining the presence or
absence of an ADA in a biological sample further comprises, after
contacting the sample with PEG, washing the precipitate.
In still other embodiments, the method further comprises
immobilizing the drug on the substrate before, after or during the
step of immobilizing the ADA on the substrate.
In another aspect, the disclosure provides a method for reducing
interference in a drug assay (e.g., a drug PK assay, a drug
quantitation assay, or a drug potency assay) due to the presence of
an ADA in a sample, the method comprising contacting the sample
with an excess amount of ADA to saturate free drug and form
drug/ADA complexes, contacting the drug/ADA complexes with
polyethylene glycol (PEG), to thereby form a precipitate comprising
drug/ADA complexes, contacting the precipitate with a solution to
dissociating the drug/ADA complexes, immobilizing the dissociated
drug on a substrate under acidic conditions, and performing the
drug assay using specific detection reagent for drug, to thereby
reduce interference from the ADA. The drug assay can be, for
example, a drug quantitation assay, a drug PK assay or a drug
potency assay.
In some embodiments, the method for reducing interference in a drug
assay due to the presence of an ADA further comprises determining
the presence or absence of, or the amount of the drug in the sample
using an anti-idiotype antibody labeled with a detectable
label.
In some embodiments, the methods disclosed herein further comprise
diluting the sample before it is contacted with an excess amount of
drug. For example, the sample is diluted 1:2, 1:5, 1:10, 1:20 fold
before it is contacted with an excess amount of drug.
In other embodiments, the sample comprises a biological sample,
wherein the biological sample comprises a material selected from
the group consisting of body fluids, mucus secretions, saliva,
blood, whole blood, plasma or serum. In some embodiments, the
sample comprises a drug.
In still other embodiments, the drug comprises an antibody or
fragment thereof, a dual affinity antibody, diabody, multiple
domain biologics (such as antibody drug conjugate), a nucleic acid
(siRNA, antisense oligonucleotide, gene therapy drugs), a peptide
or a polypeptide (native or modified), a peptidomimetic, a
carbohydrate, a lipid, or an organic or inorganic small molecule
compound, or any combinations thereof. In some embodiments, the
drug comprises a therapeutic antibody, a protein therapeutic, an
enzyme, an engineered binding protein, an engineered antibody-like
protein, a fusion protein, a scaffold protein, or any combinations
thereof. When the drug comprises an antibody or fragment thereof,
the antibody may be a murine, human, humanized or chimeric
antibody. In some embodiments, the drug is a drug modified to
exhibit less immunogenicity as compared to the same drug in
unmodified form (i.e., the drug has been modified to be less
immunogenic).
In some embodiments, the substrate comprises a carbon surface,
glass surface, silica surface, metal surface, a polymeric material,
a surface containing a metallic or chemical coating, a membrane, a
bead (e.g., a micro-bead), a porous polymer matrix, a substrate
comprising cellulosic fibers, or any combinations thereof. The
substrate can comprise a polymeric material, wherein the polymeric
material is selected from the group consisting of polystyrene,
polyvinyl chloride, polypropylene, polyethylene, polyamide, and
polycarbonate.
In some embodiments, the substrate comprises a porous carbon
surface. In one embodiment, the substrate is a high bind carbon
plate.
In some embodiments, the substrate comprises a large surface with
high coating capacity. A substrate comprising a large surface with
high coating capacity includes, for example, a high bind carbon
plate (e.g., a MSD (Meso Scale Discovery.RTM., Rockville Md.).
In some embodiments, the methods provided comprise contacting
drug/ADA complexes with polyethylene glycol (PEG) to form a
precipitate comprising drug/ADA complexes. The PEG comprises at
least one PEG compound having a molecular weight between 1,000 and
40,000 daltons, including, for example at least one PEG compound
selected from the group consisting of PEG1000, PEG1450, PEG3000,
PEG6000, PEG8000, PEG10000, PEG14000, PEG15000, PEG20000,
PEG250000, PEG30000, PEG35000, and PEG40000.
In one or more embodiments, the sample is contacted with PEG at a
concentration of between about 0.1% and about 10.0%, about 0.2% and
about 7.0%, between about 0.5% and about 6.0%, between about 0.5%
and about 5.0%, between about 1.5% and about 5.5%, between about
2.0% and about 5.0%, between about 3.0% and about 4.5%, between
about 3.5% and about 4.0%, between about 1.0% and about 2.5%,
between about 1.2% and about 1.5%, or about 0.1%, 0.2%, 0.5%, 1.0%,
1.2%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%,
6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5% or about 10.0% PEG.
In other embodiments, the methods comprise contacting a precipitate
comprising an ADA and/or ADA/drug complex with an acid solution.
The acid solution can comprise an organic acid, an inorganic acid,
or a mixture thereof. In some aspects, the acid solution comprises
an acid selected from the group consisting of citric acid,
isocitric acid, glutamic acid, acetic acid, lactic acid, formic
acid, oxalic acid, uric acid, trifluoroacetic acid, benzene
sulfonic acid, aminomethanesulfonic acid, camphor-10-sulfonic acid,
chloroacetic acid, bromoacetic acid, iodoacetic acid, propanoic
acid, butanoic acid, glyceric acid, succinic acid, malic acid,
aspartic acid, hydrochloric acid, nitric acid, phosphoric acid,
sulfuric acid, boric acid, hydrofluoric acid, hydrobromic acid and
any combinations thereof. In an exemplary embodiment, the acid
solution comprises acetic acid. For methods comprising contacting a
precipitate comprising an ADA and/or ADA/drug complex with an acid
solution, the precipitate is contacted with an acid at a
concentration of between about 0.1M to about 5M.
In other embodiments, the methods comprise contacting a precipitate
comprising an ADA and/or ADA/drug complex with a base solution. The
base solution can comprise an organic base, an inorganic base, or a
mixture thereof. In some aspects, the base solution comprises a
base selected from the group consisting of urea, sodium hydroxide,
rubidium hydroxide, cesium hydroxide, calcium hydroxide, strontium
hydroxide, barium hydroxide, zinc hydroxide, lithium hydroxide,
acetone, methylamine, and ammonia. For methods that include
contacting a precipitate comprising an ADA and/or ADA/drug complex
with a basic solution, the precipitate is contacted with a base at
a concentration of between about 0.1 M to about 1 M.
In some aspects, the disclosure provides antibodies, anti-drug
antibodies and drug labeled with (i.e., conjugated to) a detectable
label. The detectable label comprises a label selected from the
group consisting of a hapten, radioactive isotope, an enzyme, a
fluorescent label, a chemiluminescent label, and
electro-chemiluminescent label, a first member of a binding pair,
and a substrate for an enzymatic detection reaction. In one
embodiment, the detectable label comprises an
electrochemiluminescent label comprising a sulfo-TAG.RTM.
label.
In some embodiments, the detectable label comprises a fluorophore,
wherein the fluorophore is selected from the group consisting green
fluorescent protein, blue fluorescent protein, red fluorescent
protein, fluorescein, fluorescein 5-isothiocyanate (FITC), cyanine
dyes (Cy3, Cy3.5, Cy5, Cy5.5, Cy7), Bodipy dyes (Invitrogen) and/or
Alexa Fluor dyes (Invitrogen), dansyl, Dansyl Chloride (DNS-C1),
5-(iodoacetamida)fluorescein (5-IAF,
6-acryloyl-2-dimethylaminonaphthalene (acrylodan),
7-nitrobenzo-2-oxa-1,3-diazol-4-yl chloride (NBD-Cl), ethidium
bromide, Lucifer Yellow, rhodamine dyes (5-carboxyrhodamine 6G
hydrochloride, Lissamine rhodamine B sulfonyl chloride,
rhodamine-B-isothiocyanate (RITC (rhodamine-B-isothiocyanate),
rhodamine 800); tetramethylrhodamine 5-(and 6-)isothiocyanate
(TRITC)), Texas Red.TM., sulfonyl chloride, naphthalamine sulfonic
acids including but not limited to 1-anilinonaphthalene-8-sulfonic
acid (ANS) and 6-(p-toluidinyl)naphthalen-e-2-sulfonic acid (TNS),
Anthroyl fatty acid, DPH, Parinaric acid, TMA-DPH, Fluorenyl fatty
acid, Fluorescein-phosphatidylethanolamine, Texas
red-phosphatidylethanolamine, Pyrenyl-phophatidylcholine,
Fluorenyl-phosphotidylcholine, Merocyanine 540, Naphtyl Styryl,
3,3'dipropylthiadicarbocyanine (diS-C3-(5)), 4-(p-dipentyl
aminostyryl)-1-methylpyridinium (di-5-ASP), Cy-3 lodo Acetamide,
Cy-5-N-Hydroxysuccinimide, Cy-7-Isothiocyanate, IR-125, Thiazole
Orange, Azure B, Nile Blue, Al Phthalocyanine, Oxaxine
1,4',6-diamidino-2-phenylindole. (DAPI), Hoechst 33342, TOTO,
Acridine Orange, Ethidium Homodimer,
N(ethoxycarbonylmethyl)-6-methoxyquinolinium (MQAE), Fura-2,
Calcium Green, Carboxy SNARF-6, BAPTA, coumarin, phytofiuors and
Coronene.
In some embodiments, the detectable label comprises an enzyme that
catalyzes a color change reaction, including, an enzyme selected
from the group consisting of alkaline phosphatase,
beta-galactosidase, horse radish peroxidase, urease and
beta-lactamase and glucose oxidase.
In some embodiments, the detectable label comprises a first member
of a binding pair or a second member of a binding pair, wherein the
binding pair is selected from the group consisting of
biotin/streptavidin, biotin/avidin, biotin/neutravidin,
biotin/captavidin, epitope/antibody, protein A/immunoglobulin,
protein G/immunoglobulin, protein L/immunoglobulin,
GST/glutathione, His-tag/Nickel, antigen/antibody, FLAG/M1
antibody, maltose binding protein/maltose, calmodulin binding
protein/calmodulin, enzyme-enzyme substrate, and receptor-ligand
binding pairs.
In some embodiments, the detectable label comprises a first member
of a binding pair; and the second member of the binding pair is
conjugated to an enzyme, an antibody epitope, an antigen, a
fluorophore, a radioisotope, a nanoparticle, a member of a second
binding pair, and a metal chelate.
In other embodiments, the detectable label comprises a first member
of a binding pair, wherein the first member of the binding pair is
biotin and the second member of the binding pair is selected from
the group consisting of streptavidin, avidin, neutravidin and
capravidin, and the second member of the binding pair conjugated to
an enzyme.
The methods provided herein can be performed on either a manual or
automated instrument platform, depending on the number of samples
to be tested.
"Anti-drug antibodies" or "ADAs" are antibodies that bind
specifically to any region of a drug. For example, an anti-drug
antibody may be an antibody or fragment thereof, which may be
directed against any region of a drug antibody, e.g., the variable
domain, the constant domains, or the glycostructure of the
antibody). Such anti-drug antibodies may occur during drug therapy
as an immunogenic reaction of a patient. An ADA may be one of any
human immunoglobulin isotype (e.g., IgM, IgE, IgA, IgG, IgD) or IgG
subclass (IgG1, 2, 3, and 4). ADAs include ADAs from any animal
source, including, for example, human or non-human animal (e.g.
veterinary) sources.
For the purpose of the present specification, the term "NAb" or
"neutralizing antibody" refers to an antibody that binds to an
endogenously produced molecule, e.g., an antibody, nucleic acid,
peptide, polypeptide, peptidomimetic, carbohydrate or lipid. For
example, a NAb may be an endogenously produced protein, such as,
for example, erythropoietin or insulin. The NAb may or may not
reduce (e.g., neutralizes) at least one biological activity of the
endogenously produced molecule.
For instance, in some aspects, the disclosure provides a method for
determining the presence or absence of NAb in a sample, the method
comprising contacting the sample with an excess amount of antigen
to which the NAb binds to form an antigen/Nab complexes, contacting
the antigen/NAb complexes with polyethylene glycol (PEG), to form a
precipitate comprising antigen/NAb complexes, contacting the
precipitate with a solution to dissociate the antigen/NAb
complexes, immobilizing the dissociated NAbs on a surface and/or
substrate, and determining the presence of or amount of said NAb.
In a further aspect, the determining step comprises contacting the
immobilized NAb with of an antigen to which the Nab binds
conjugated with a detectable label, and determining the presence of
or amount of said detectable label, to thereby determine the
presence or amount (titer) of NAb in the sample.
In the context of the invention, the term "patient" refers to any
subject, preferably a mammal, and more preferably a human, with a
disease or suspected of having a disease. The term "subject," as
used herein, refers to any animal (e.g., a human or non-human
animal subject). In some instances, the subject is a mammal. In
some instances, the term "subject", as used herein, refers to a
human (e.g., a man, a woman, or a child). In some instances, the
term "subject", as used herein, refers to laboratory animal of an
animal model study.
As used herein, the term "biological sample" or "sample" refers to
a sample obtained or derived from a patient which comprises patient
derived immunoglobulin and may therefore be referred to as an
immunoglobulin sample. By way of example, a biological sample
comprises a material selected from the group consisting of body
fluids, blood, whole blood, plasma, serum, mucus secretions,
saliva, cerebrospinal fluid (CSF), bronchioalveolar lavage fluid
(BALF), fluids of the eye (e.g., vitreous fluid, aqueous humor),
lymph fluid, lymph node tissue, spleen tissue, bone marrow, and an
immunoglobulin enriched fraction derived from one or more of these
tissues. In some embodiments the sample is, or comprises blood
serum or is an immunoglobulin enriched fraction derived from blood
serum or blood. The sample is, or can be derived (obtained) from, a
bodily fluid or body tissue. In some embodiments, the sample is
obtained from a subject who has been exposed to the drug, such as
repeatedly exposed to the same drug. In other embodiments, the
sample is obtained from a subject who has not recently been exposed
to the drug, or obtained from the subject prior to the planned
administration of the drug.
The term "substrate", as used herein refers to any material or
macromolecular complex to which an ADA or drug material (e.g., an
antibody, nucleic acid, peptide, polypeptide, peptidomimetic,
carbohydrate, lipid, or an organic or inorganic small molecule
compound) may bind. The composition and/or surface of the substrate
should allow for binding of an ADA or drug material under acidic
conditions (or basic conditions) that allow for dissociation of the
ADA/drug complexes. In some embodiments, these substrates have a
high loading capacity, which improves sensitivity, thus allowing
for detection of ADAs and/or drug materials present in relatively
low concentrations. Examples of commonly used substrates include,
but are not limited to, carbon surfaces (e.g. a porous or high bind
carbon plate), glass surfaces, silica surfaces, plastic surfaces,
metal surfaces, surfaces containing a metallic or chemical coating,
membranes (e.g., nylon, polysulfone, silica), micro-beads (e.g.,
latex, polystyrene, or other polymer), porous polymer matrices
(e.g., polyacrylamide gel, polysaccharide, polymethacrylate), and
substrates comprising cellulosic fibers (e.g., cellulose sponges,
cellulose paper). In one aspect, the porous or high bind carbon
plate is a MSD (Meso Scale Discovery.RTM.) high bind plate. The
substrate may be a biosensor chip, microarray, or lab-on-chip
capable of sensing a target molecule. Any kind of biosensor that is
capable of sensing specific binding to the biosensor chip is
applicable, including commercially available biosensors, such as
the biosensors produced by Biacore.
As used herein, an entity (e.g., antibody, anti-drug antibody,
drug, protein, enzyme, antibody, antibody fragment, multiple domain
biotherapeutics (e.g., antibody drug conjugates), or related
species) that is modified by the term "labeled" includes any entity
that is conjugated with another molecule or chemical entity a that
is empirically detectable (e.g., "detectable label"). Chemical
species suitable as labels for labeled-entities include, but are
not limited to, enzymes, fluorescent dyes; quantum dots; optical
dyes; luminescent dyes; and radionuclides.
As used herein, the term "one or more" includes at least one, more
suitably, one, two, three, four, five, ten, twenty, fifty,
one-hundred, five-hundred, etc., of the item to which "one or more"
refers.
The approach disclosed herein has been shown to eliminate drug
interference in ADA assays. In practice, this method principle can
be applied to reduce/eliminate the interferences in any type of
immunoassay. This method principle can also be used for any ligand
binding assays for ADA, PK and biomarkers. The methods described
herein can be applied to ligand binding assays to test for
neutralizing antibodies (NAbs). The ligand binding assays can
include competitive inhibition of drug binding to drug target. In
PK and biomarker assays, excess antibody can be added for complex
formation and after precipitation and acid dissociation; detection
is made using labeled detection antibody. In all cases, it is
important to optimize the concentration of PEG in the assay to
balance the sensitivity and specificity. The higher concentration
of PEG, the lower molecular weight protein it will precipitate.
Therefore, to specifically precipitate desired complex containing
target analyte (such as antibody-drug complex precipitation), one
needs to minimize the amount on unbound non-specific proteins to be
precipitated (such as serum IgM and IgG). The MSD high bind plate
was utilized in the studies due to its carbon and porous structure.
Based on assay design principle, other large capacity coating
surfaces would also work.
Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control.
Other features and advantages of the invention will be apparent
from the following detailed description and figures, and from the
claims.
DESCRIPTION OF DRAWINGS
FIG. 1 is a schematic representation of an embodiment of an ADA
assay according to the invention.
FIGS. 2A and 2B are graphs depicting the results of an example
bridging assay format detecting affinity purified rabbit antibody
at levels ranging from 8 .mu.g/mL to 125 ng/mL with various
concentration of Drug A (0, 1, 10 and 100 .mu.g/mL) tested in the
MSD bridging assay format without acid dissociation. A. The
observed S/B was plotted against the ADA concentration to assess
the recovery of the antibody with different levels of drug compared
to baseline without drug. B. Percent recovery relative to baseline.
ADA: MSD bridging assay: Meso Scale Discovery.RTM. bridging; S/B:
Signal-to-background.
FIGS. 3A and 3B are graphs depicting the results of an example
bridging assay format detecting affinity purified rabbit antibody
at levels ranging from 8 .mu.g/mL to 125 ng/mL with various
concentration of Drug A (0, 1, 10 and 100 .mu.g/mL) tested in the
MSD bridging assay format with acid dissociation. A. The observed
S/B was plotted against the ADA concentration to assess the
recovery of the antibody with different levels of drug compared to
baseline without drug. B. Percent recovery relative to baseline.
S/B: Signal-to-background.
FIGS. 4A and 4B are graphs depicting the results of an example
bridging assay format detecting affinity purified rabbit antibody
at levels ranging from 8 .mu.g/mL to 125 ng/mL with various
concentration of Drug A (0, 1, 10 and 100 .mu.g/mL) tested in the
MSD bridging assay using the polyethylene glycol precipitation and
acid dissociation assay format of the invention (i.e., the PandA
assay format). A. The observed S/B was plotted against the ADA
concentration to assess the recovery of the antibody with different
levels of drug compared to baseline without drug. B. Percent
recovery relative to baseline. S/B: Signal-to-background.
FIG. 5 is a graph depicting the results of an example bridging
assay format detecting affinity purified rabbit anti-Drug A
antibody with no drug tested in the PEG and Acid (PandA) assay
format using three different lots of MSD high bind plates. The
observed S/B was plotted against the ADA concentration. ADA:
Anti-drug antibody; S/B: Signal-to-background.
FIG. 6 is a graph depicting the results of an example bridging
assay format detecting Drug B ADAs in pooled normal serum samples
(n=32) evaluated in the MSDB assay format with and without acid
dissociation. Normal Population Distribution (Drug B bridging
assays). No treatment=without dissociation; Acid Treated=with acid
dissociation; S/B: Signal-to-background.
FIG. 7 is a graph depicting the results of an example bridging
assay format detecting Drug B ADAs in pooled disease baseline serum
samples (n=16) evaluated in the MSD bridging assay format with and
without acid dissociation, as well as the PandA assay format to
determine population distribution.
FIGS. 8A and 8B are graphs depicting the results of an example
bridging assay format detecting affinity purified rabbit antibody
at levels ranging from 4 .mu.g/mL to 31.3 ng/mL with various
concentration of Drug B (0, 10, 50 and 250 .mu.g/mL) tested in the
MSD bridging assay format without acid dissociation. A. The
observed S/B was plotted against the ADA concentration to assess
the recovery of the antibody with different levels of drug compared
to baseline without drug. B. Percent recovery relative to baseline.
S/B: Signal-to-background.
FIGS. 9A and 9B are graphs depicting the results of an example
bridging assay format detecting affinity purified rabbit antibody
at levels ranging from 4 .mu.g/mL to 31.3 ng/mL with various
concentration of drug B (0, 10, 50 and 250 .mu.g/mL) tested in the
MSD bridging assay using the PandA assay format. A. The observed
S/B was plotted against the ADA concentration to assess the
recovery of the antibody with different levels of drug compared to
baseline without drug. B. Percent recovery relative to baseline.
S/B: Signal-to-background.
FIGS. 10A and 10B are graphs depicting the results of an example
bridging assay format detecting affinity purified rabbit antibody
at levels ranging from 8 .mu.g/mL to 125 ng/mL with various
concentration of Drug C (0, 2.5, 25 and 250 .mu.g/mL) tested in the
MSD bridging assay format with acid dissociation. A. The observed
S/B was plotted against the ADA concentration to assess the
recovery of the antibody with different levels of drug compared to
baseline without drug. B. Percent recovery relative to baseline.
S/B: Signal-to-background.
FIGS. 11A and 11B are graphs depicting the results of an example
bridging assay format detecting affinity purified rabbit antibody
at levels ranging from 8 .mu.g/mL to 125 ng/mL with various
concentration of Drug C (0, 2.5, 25 and 250 .mu.g/mL) evaluated in
the MSD bridging assay using the PandA assay format. A. The
observed S/B was plotted against the ADA concentration to assess
the recovery of the antibody with different levels of drug compared
to baseline without drug. B. Percent recovery relative to baseline.
S/B: Signal-to-background.
DETAILED DESCRIPTION
The present inventors have developed a novel approach for
qualitatively and/or quantitatively detecting ADAs from a sample
which is effective in reducing and eliminating the interference
problems caused by drug or target in ADA detection. Using the
principle of PEG precipitation, acid dissociation, coating on high
capacity surface under acidic condition, the methods described
herein allow for specific detection of ADA as well as drug or drug
target using specific detection reagent. The approach can be
applied to broader applications for reduction or elimination of
interference in immunoassays for ADA, PK/TK, and biomarker (such as
drug target) assays, as well as ligand-binding assays for detection
of neutralizing antibodies.
For a drug with a long half-life and/or one administered at a high
dose or a repeated dose, such as an antibody-based therapy, the ADA
usually forms circulating immune complexes with the drug, typically
making the ADA unavailable for detection. For example, circulating
drug may interfere with the detection of ADAs and drug target, or
ADAs may interfere with the detection and/or accurate quantitation
of drug levels for pharmacokinetic ("PK") studies and or
toxicokinetics ("TK") studies. Monoclonal antibody drug
interference, especially from human IgG4 drugs, presents an
additional challenge for ADA analysis due to its longer half-life
and higher doses. The impact of such interference is specific to
the immunoassay method and platform and may be dependent on the
reagents used in each respective assay. Development of drug
tolerant immunogenicity assays becomes more challenging when the
drug itself is a humanized antibody therapeutic.
The most widely adopted approaches in use currently still have
limitations of timing, sensitivity or accuracy due to the presence
of interference factors that are the same or resemble the binding
partners in the ligand assay. These interferences include but not
limited to drug interference in the ADA assay, ADA and/or drug
target interference in the PK assay, drug interference in the drug
target biomarker assay, etc. The commonly used ADA method is the
bridging assay where a multi-valent ADA bridges between a capture
drug (unlabeled or biotin labeled) and a labeled detection drug.
This format is susceptible to endogenous drug interference (false
negative ADA) and/or drug target interference (false positive ADA).
Being recognized as one of the major challenges in the analytical
method development field, many approaches have been used to
mitigate this problem such as acid or base dissociation, 3rd party
binding partner competitive inhibition, and/or removal of the
interference factors, solid phase extraction (SPEAD), ACE and many
others. The use of acid dissociation in sample treatment in
conjunction with a bridging assay allows for some improvement in
the detection of ADAs in the presence of a drug with improved assay
sensitivity and ADA recovery. The same is typically observed with
the SPEAD and ACE which usually allow for some improvement in drug
tolerance; but sensitivity and relative accuracy is not fully
maintained. Although acid (or base) dissociate the ADA/drug immune
complex, once the mixture is neutralized under the assay condition,
the immune complexes re-form. In currently used ADA assays, ADA
detection is only possible if the production of ADA exceeds the
amount of drug present in the patient's serum due to the formation
of ADA-drug complexes. Such drug interference leads to an
underestimation of the number of patients of patients producing
ADA.
Immunogenicity of drug products, particularly therapeutic proteins,
is a major concern in clinical and preclinical studies since it can
lead to potentially serious side effects, loss of efficacy, and
changes in drug exposure, complicating the interpretation of
toxicity, pharmacokinetic (PK) and pharmacodynamics (PD) data. As
the number of drug products with long half-life such as monoclonal
antibodies is increasing, drug tolerance in ADA assays is of
growing concern. To assess the overall immunogenic potential of a
drug, the total amount of ADA is of particular interest. Many
techniques that are widely used for detection of ADAs in serum and
plasma depend on the specific binding of the ADA to its target drug
via the antigen binding site. For example, bridging assay formats
rely on the availability of the antigen binding sites on ADAs. As
these many assay formats rely on the availability of the antigen
binding sites on the ADAs, only drug-free or partially drug-free
ADA can be detected. Because specific assay materials are sparse
and time is pressing, an assay format with improved drug tolerance
for detection of ADAs in biological samples is highly desirable
Drug tolerance is generally defined as the maximal amount of free
drug in a sample that still results in a detectable ADA signal.
Acid treatment of samples has been used to improve free drug
tolerance in ADA assays. Antibody-antigen (or drug) binding is
weakened and eventually disrupted by low pH, making detection of
free ADA that is dissociated from partially or completely
drug-bound ADAs possible in many immunogenic assay formats (i.e.,
bridging assay formats), thereby improving drug tolerance. As
demonstrated in the examples provided herein, however, acid
treatment alone does not eliminate drug tolerance in ADA
assays.
In some embodiments, antibody-antigen (or drug) binding is weakened
and eventually disrupted by high pH, making detection of free ADA
that is dissociated from partially or completely drub-bound ADAs,
following treatment with a basic solution, possible in many
immunogenic assay formats. The structural characteristics of the
biologic drug, such as pI, or the presence of certain conjugating
bonds, will dictate whether an acid solution or basic solution is
more appropriate for disrupting ADA/drug binding. To increase drug
tolerance and provide an improved ADA assay format, the present
inventors have developed methods for determining the presence or
absence of an ADA in a sample with improved drug tolerance. The
methods contemplated herein include a polyethylene glycol
precipitation step and an acid dissociation step.
As disclosed herein, the present invention relates to a method for
determining the presence or absence of an ADA in a sample (e.g., a
biological sample), the method comprising contacting the sample
with an excess amount of drug to which the ADA binds to form
drug/ADA complexes, contacting the drug/ADA complexes with
polyethylene glycol (PEG), to form a precipitate comprising
drug/ADA complexes, contacting the precipitate with a solution to
dissociate the drug/ADA complexes, immobilizing the dissociated
ADAs on a substrate, and determining the presence of or amount of
said ADA. In a further aspect, the determining step comprises
contacting the immobilized ADAs with drug conjugated to a
detectable label and determining the presence of or amount of said
detectable label, to thereby determine the presence or absence of
ADA in the sample.
In one embodiment, the precipitate (drug/ADA complexes) is
contacted with an acid solution to dissociate the complexes and
then coated on a large surface (under acidic conditions to keep
drug and ADA apart) or substrate (e.g., a high bind carbon plate
with high coating capacity) for a time sufficient to allow coating
of all dissociated free drugs and free ADAs. The acidic environment
prevents the complexes from reforming while being immobilized onto
the substrate surface. When an acid solution is used to dissociate
the complexes, the assay can be referred to as a PandA (PEG and
Acid) assay.
In another embodiment, the final precipitate (drug/ADA complexes)
is reconstituted with a basic solution to dissociate the complexes
and then coated on a large surface (under basic conditions to keep
drug and ADA apart) or substrate (e.g., a high bind carbon plate
with high coating capacity) for a time sufficient to allow coating
of all dissociated free drugs and free ADAs. The basic environment
prevents the complexes from reforming while being immobilized onto
the substrate surface.
The selection of an acidic or basic solution will depend on the
parameters of the drug (e.g., the biologic drug), such as pI, or
the presence of certain conjugating bonds, and the selection will
have minimal effect on the integrity and structure of the drug.
The present invention also relates to methods for reducing
interference in a drug PK/TK assay due to the presence of an ADA in
a biological sample, the method comprising contacting the sample
with an excess amount of an ADA to form drug/ADA complexes,
contacting the drug/ADA complexes with polyethylene glycol (PEG),
to thereby form a precipitate comprising drug/ADA complexes,
contacting the precipitate with an acid solution, thereby
dissociating the drug/ADA complexes, immobilizing the dissociated
free drug and free ADA on a surface and/or substrate, and
performing the drug PK/TK assay using specific detection reagent
against drug, to thereby reduce interference from the ADA. The drug
assay can be, for example, a drug quantitation assay, a drug PK/TK
assay or a drug potency assay. Similar principle can be applied to
method for quantitation of drug target as biomarker assay for drug
safety and efficacy (PD) in the presence of drug interference.
Excess drug is added to the sample to form drug target/drug
complex. PEG precipitation and acid dissociation (or base
dissociation) steps are used and specific drug target detection
reagent is then used.
In some aspects the disclosure provides methods for determining the
presence or absence of an ADA directed against a drug. The term
"drug" or "drug material", as used herein refers to a chemical that
has medicinal, performance-enhancing, and/or intoxicating effects
when introduced into the body of a human or other animal. For
example, the drug can be an organic or inorganic small molecule
compound or a biologic therapeutic (e.g., an antibody (e.g., a drug
antibody) or fragment thereof, multiple domain biotherapeutics,
nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate,
or lipid), so long as the drug is immunogenic and capable of
eliciting an immune response. The term "drug antibody" denotes an
antibody which can be administered to an individual for the
treatment of a disease and as used herein distinguishes such
antibodies from ADAs. Non-limiting examples of drug antibodies
include, for example, an antibody selected from muromomab-CD3,
abciximab, rituximab, daclizumab, basiliximab, palivizumab,
infliximab, trastuzumab, etanercept, gemtuzumab, fresolimumab,
alemtuzumab, ibritomomab, adalimumab, alefacept, omalizumab,
tofacitinib, tositumomab, efalizumab, cetuximab, bevacizumab,
natalizumab, ranibizumab, panitumumab, eculizumab mepolizumab,
necitumumab, blinatumomab, nivolumab, dinutuximab, secukinumab,
evolocumab, pembrolizumab, ramucirumab, vedoluzumab, siltuximab,
opinutuzumab, adotrastuzumab emtansine, raxibacumab, pertuzumab,
brentuximab, belimumab, ipilimumab, denosumab, tocilizumab,
ofatumumab, canakinumab, golimumab, ustekinumab, catumaxomab, and
certolizumab.
The methods disclosed herein may comprise a polyethylene glycol
("PEG") mediated precipitation step comprising contacting a sample
with a PEG compound. The PEG compound may be a PEG compound having
a molecular weight between 1,000 and 40,000 daltons. For example,
the PEG compound comprises at least one PEG selected from the group
consisting of selected from the group consisting of PEG1000,
PEG1450, PEG3000, PEG6000, PEG8000, PEG10000, PEG14000, PEG15000,
PEG20000, PEG250000, PEG30000, PEG35000, and PEG40000.
The specific concentration of PEG is selected to maximize the
balance of specificity and selectivity. The amount of PEG contacted
with the sample may correspond to a concentration of between about
0.1% and about 10.0%, about 0.2% and about 7.0%, between about 0.5%
and about 6.0%, between about 0.5% and about 5.0%, between about
1.5% and about 5.5%, between about 2.0% and about 5.0%, between
about 3.0% and about 4.5%, between about 3.5% and about 4.0%,
between about 1.0% and about 2.5%, between about 1.2% and about
1.5%, or about 0.1%, 0.2%, 0.5%, 1.0%, 1.2%, 1.5%, 2.0%, 2.5%,
3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%,
8.5%, 9.0%, 9.5% or about 10.0% PEG.
The methods may comprise an acid dissociation step comprising
contacting a precipitate with an acid. The acid may be or include
an organic acid. Alternatively or in addition, the acid may be or
include an inorganic acid. The acid used in the dissociation step
may comprise a mixture of an organic acid and an inorganic acid.
Non-limiting examples of organic acids include, for example, citric
acid, isocitric acid, glutamic acid, acetic acid, lactic acid,
formic acid, oxalic acid, uric acid, trifluoroacetic acid, benzene
sulfonic acid, aminomethanesulfonic acid, camphor-10-sulfonic acid,
chloroacetic acid, bromoacetic acid, iodoacetic acid, propanoic
acid, butanoic acid, glyceric acid, succinic acid, malic acid,
aspartic acid, and combinations thereof. Non-limiting examples of
inorganic acids include, for example, hydrochloric acid, nitric
acid, phosphoric acid, sulfuric acid, boric acid, hydrofluoric
acid, hydrobromic acid, and mixtures thereof.
The amount of an acid may correspond to a concentration of between
about 0.01M to about 10M, between about 0.1M to about 5M, about
0.1M to about 2M, between about 0.2M to about 1M, or between about
0.25M to about 0.75M of an acid or a mixture of acids. In some
instances the amount of an acid corresponds to a concentration of
greater than or equal to about 0.01M, 0.05M, 0.1M, 0.2M, 0.3M,
0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 2M, 3M, 4M, 5M, 6M, 7M, 8M,
9M, or 10M of an acid or a mixture of acids. The pH of the acid can
be, for example, about 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,
4.5, 5.0, 5.5, 6.0, or 6.5.
The methods may comprise an base dissociation step comprising
contacting a precipitate with an base. The base may be or include
an organic base. Alternatively or in addition, the acid may be or
include an inorganic base. The base used in the dissociation step
may comprise a mixture of an organic base and an inorganic base.
Non-limiting examples of bases include, for example, urea, sodium
hydroxide, rubidium hydroxide, cesium hydroxide, calcium hydroxide,
strontium hydroxide, barium hydroxide, zinc hydroxide, lithium
hydroxide, acetone, methylamine, and ammonia, and mixtures
thereof.
Where a basic solution is used to disrupt the ADA/drug interaction,
the amount of base may correspond to a concentration of between
about 0.01M to about 5M, between about 0.1M to about 5M, about 0.1M
to about 1M, between about 0.2M to about 1M, or between about 0.25M
to about 0.75M of a base or a mixture of bases. In some instances
the amount of a base corresponds to a concentration of greater than
or equal to about 0.01M, 0.05M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M,
0.7M, 0.8M, 0.9M, 1M, 2M, 3M, 4M, 5M, 6M, 7M, 8M, 9M, or 10M of a
base or a mixture of bases. The pH of the base can be, for example,
about 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5 and
13.0.
In some embodiments, the sample is contacted with an acid or base
for an amount of time sufficient to dissociate preformed drug/ADA
complexes. In certain instances, the sample is contacted (e.g.,
incubated) with an acid or base for a period of time ranging from
about 0.1 hours to about 24 hours, e.g., about 0.2 hours to about
16 hours, about 0.5 hours to about 10 hours, about 0.5 hours to
about 5 hours, or about 0.5 hours to about 2 hours. In other
instances, the sample is contacted (e.g., incubated) with an acid
or base for a period of time that is greater than or equal to about
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3,
3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 hours. The sample can be
contacted with an acid or a base at any temperature that is
generally compatible with the method, e.g., 4.degree. C., room
temperature (RT), or 37.degree. C. RT can be, for example
22.degree. C. to 26.degree. C., e.g., 23.degree. C., 24.degree. C.
or 25.degree. C.
The methods may comprise immobilizing a dissociated ADA and/or drug
on a surface and/or substrate. The substrate may comprise a carbon
surface, glass surface, silica surface, metal surface, a surface
coated with a polymeric material, a surface containing a metallic
or chemical coating, a membrane, micro-beads, or a porous polymer
matrix. The substrate may comprise a porous carbon surface. In an
exemplary embodiment, the substrate is a high bind carbon plate
having a large surface and high coating capacity.
The methods may further comprise determining the presence of or
amount of an ADA, or the presence of or amount of a drug in the
sample. Thus, the disclosure provides antibodies, anti-drug
antibodies or drug labeled with a detectable label. Non-limiting
examples of detectable labels for any of the methods of the
invention include a hapten, an enzyme, an enzyme substrate, an
enzyme inhibitors, a fluorophore, a chromophores, luminescent
markers, radioisotopes (including radionucleotides) and a member of
a binding pair. The intensity of the detectable label can be
measured using instruments and devices known to those skilled in
the art, including, for example a portable or benchtop fluorometer
(e.g., a handheld fluorometer) or a portable or benchtop
colorimeter (e.g., a handheld colorimeter).
In some embodiments, the detectable label is an
electrochemiluminescent label, including, for example, a
Sulfo-TAG.RTM. label.
The detectable label can be a specific member (a first member or a
second member) of a binding pair. Binding pairs for use in the
methods provided herein include, for example, biotin/streptavidin,
biotin/avidin, biotin/neutravidin, biotin/captavidin,
epitope/antibody, protein A/immunoglobulin, protein
G/immunoglobulin, protein L/immunoglobulin, GST/glutathione,
His-tag/Nickel, antigen/antibody, FLAG/M1 antibody, maltose binding
protein/maltose, calmodulin binding protein/calmodulin,
enzyme-enzyme substrate, and receptor-ligand binding pairs. In some
embodiments, the GlcNac binding protein is conjugated to a first
member of binding pair (e.g., biotin, avidin, neutravidn, captavid,
antibody, antigen, protein A, protein G, protein L, GST, His-Tag,
FLAG, MBP, calmodulin binding protein, an enzyme, a receptor or
ligand).
As used herein, the terms "fluorescence label" and "fluorophore"
used interchangeably and refer to any substance that emits
electromagnetic energy such as light at a certain wavelength
(emission wavelength) when the substance is illuminated by
radiation of a different wavelength (excitation wavelength) and is
intended to encompass a chemical or biochemical molecule or
fragments thereof that is capable of interacting or reacting
specifically with an analyte of interest in a sample to provide one
or more optical signals.
Representative fluorophores for use in the methods provided herein
include, for example, green fluorescent protein, blue fluorescent
protein, red fluorescent protein, fluorescein, fluorescein
5-isothiocyanate (FITC), cyanine dyes (Cy3, Cy3.5, Cy5, Cy5.5,
Cy7), Bodipy dyes (Invitrogen) and/or Alexa Fluor dyes
(Invitrogen), dansyl, Dansyl Chloride (DNS-C1),
5-(iodoacetamida)fluorescein (5-IAF,
6-acryloyl-2-dimethylaminonaphthalene (acrylodan),
7-nitrobenzo-2-oxa-1,3-diazol-4-yl chloride (NBD-Cl), ethidium
bromide, Lucifer Yellow, rhodamine dyes (5-carboxyrhodamine 6G
hydrochloride, Lissamine rhodamine B sulfonyl chloride,
rhodamine-B-isothiocyanate (RITC (rhodamine-B-isothiocyanate),
rhodamine 800); tetramethylrhodamine 5-(and 6-) isothiocyanate
(TRITC)), Texas Red.TM., sulfonyl chloride, naphthalamine sulfonic
acids including but not limited to 1-anilinonaphthalene-8-sulfonic
acid (ANS) and 6-(p-toluidinyl)naphthalen-e-2-sulfonic acid (TNS),
Anthroyl fatty acid, DPH, Parinaric acid, TMA-DPH, Fluorenyl fatty
acid, Fluorescein-phosphatidylethanolamine, Texas
red-phosphatidylethanolamine, Pyrenyl-phophatidylcholine,
Fluorenyl-phosphotidylcholine, Merocyanine 540, Naphtyl Styryl,
3,3'dipropylthiadicarbocyanine (diS-C3-(5)), 4-(p-dipentyl
aminostyryl)-1-methylpyridinium (di-5-ASP), Cy-3 lodo Acetamide,
Cy-5-N-Hydroxysuccinimide, Cy-7-Isothiocyanate, IR-125, Thiazole
Orange, Azure B, Nile Blue, Al Phthalocyanine, Oxaxine
1,4',6-diamidino-2-phenylindole. (DAPI), Hoechst 33342, TOTO,
Acridine Orange, Ethidium Homodimer,
N(ethoxycarbonylmethyl)-6-methoxyquinolinium (MQAE), Fura-2,
Calcium Green, Carboxy SNARF-6, BAPTA, coumarin, phytofiuors,
Coronene, and metal-ligand complexes.
Haptens for use in the methods provided herein include, for
example, digoxigenin, and biotin.
Enzymes for use in the methods provided herein include, for
example, alkaline phosphatase (AP), beta-galactosidase, horse
radish peroxidase (HRP), soy bean peroxidase (SBP), urease,
beta-lactamase and glucose oxidase.
In certain aspects, this disclosure provides kits that are adapted
for determining the presence or absence of an ADA in a biological
sample. The kits may comprise instructions and, in a container,
reagents for contacting drug/ADA complexes with polyethylene glycol
(PEG), to form a precipitate comprising drug/ADA complexes and
reagents for contacting the precipitate with an acid solution to
dissociating the drug/ADA complexes; and a substrate suitable for
immobilizing the dissociated ADAs or drug on for further
analysis.
Examples
The invention is further described in the following examples, which
do not limit the scope of the invention described in the
claims.
The examples below describe novel ADA assay methods where complete
recovery of an antibody was obtained at the limit of quantitation
despite the presence of high levels of the antibody therapeutic.
Specifically, three case studies are provided to demonstrate
elimination of drug interference in ADA assays for the monoclonal
antibody therapeutics (drugs A, B and C).
1.1 Assay Format
To improve the drug tolerance (or eliminate drug interference) of
conventional immunogenicity assays, the inventors sought to develop
a new method for determining the presence or amount of an ADA in a
sample (e.g., a biological sample). The inventors have successfully
developed a new method where complete recovery of anti-drug
antibody was obtained at the limit of quantitation despite the
presence of high levels of drug (the antibody therapeutic). A
schematic representation of an exemplary embodiment of the method
is shown in FIG. 1.
As depicted in FIG. 1, excess drug material is added to a sample
(e.g., a biological sample) to allow drug/ADA complexes to form.
Following the initial incubation, PEG is added to each sample and
incubated to allow for precipitation of complexes. After a series
(e.g., one or more) of washes, the precipitate is reconstituted
with an acid solution to dissociate drug/ADA complexes. The
dissociated drug/ADA complexes are coated on a substrate (i.e.,
coated on the ells of a high bind MSD plate). Following incubation,
the substrate is blocked and then detection is performed using drug
labeled with a detectable label allowing for ADA detection by ECL
("electrochemiluminescence) or "enhanced chemiluminescence").
In one aspect, the method depicted in FIG. 1 comprises adding
excess drug material to the samples to saturate free antibody
therefore forming drug/ADA complexes. The complexes are then
precipitated using PEG. PEG is added to the sample at a
concentration optimized to achieve the desired sensitivity while
maintaining specificity. After a series of washes, the final
precipitate is reconstituted with an acid solution and coated on a
high bind carbon plate. The acidic environment prevents the
complexes from reforming while being absorbed onto the porous
carbon surface of the MSD high bind plate. Detection of the total
ADA levels is then performed using Sulfo-TAG.RTM. label conjugated
drug followed by Electrochemiluminescence read out on a Meso Scale
Discovery.RTM. Sector 2400 reader.
1.2 Experimental Materials
Therapeutic monoclonal antibodies, Sulfo-TAG.RTM. drug, and
affinity purified rabbit anti-drug are developed by Genzyme, a
Sanofi Company (Framingham, Mass.). Naive human serum pools from
healthy individuals were purchased from Bioreclamation Inc. Disease
baseline serum samples were obtained from treatment naive subjects
enrolled in product-related clinical trials. High Bind 96-well Meso
Scale plates, Sulfo Tag, read Buffer T, Sector 2400 reader attained
from Meso Scale Discovery.RTM. ("MSD"). PEG8000 was obtained from
TekNova. Glacial acetic acid was provided by J. T Baker. Tween 20
and Non-Fat Dry Milk-Sigma, acquired from Aldrich. The ELx405 plate
washer was supplied by Biotek. Plate wash buffer came from
PerkinElmer. Clear polypropylene 96-well microtiter plates were
procured from Corning. Bovine Serum Albumin was obtained from
Seracare. Borate is found through Sigma Aldrich, Catalog number
B0394.
Drug A is a humanized IgG1 depleting antibody that binds to
lymphocyte cell surface target for an autoimmune disease. Drug B is
a full human IgG4 that neutralizes a soluble cytokine binding to
its cell surface receptor in the target tissue for a fibrosis
indication. Drug C is a humanized IgG4 that recognizes cell surface
adhesion molecule and blocks its binding to a soluble ligand.
1.3 Bridging Immunoassay without Acid Dissociation Procedure
The MSD bridging assay format requires the drug to be labeled with
biotin and labeled with sulfo-TAG.RTM.. According to the MSD assay
format, the biotinylated drug will serve as the capture molecule
and the sulfo-TAG.RTM. labeled drug will be the reporter in the
bridging assay.
Samples are initially diluted 1:10 in assay buffer ((300 mM Acetic
acid, 2% BSA). Samples are added to a polypropylene plate in
duplicate wells and a solution containing equi-molar concentrations
of Biotin-Drug and Sulfo-TAG-Drug in assay buffer is added. The
plate is then incubated for 2 hours in a 22-26.degree. C. shaker at
450 rpm. A streptavidin coated MSD plate is blocked with assay
buffer for a minimum of one hour. Following the incubation, the MSD
plate is washed 3 times and 50 .mu.L of the incubated mixture is
transferred from the polypropylene plate to the MSD plate and
incubated for 2 hours on a 22-26.degree. C. shaker. Following the
incubation, the plate is washed and a solution of MSD read buffer T
containing tripropylamine is added and the plate is read on the MSD
sector imager 2400.
Within the instrument, a voltage is applied. In the presence of
tripropylamine, sulfo-TAG.RTM. participates in an
electro-chemiluminescent (ECL) reaction. The antibodies bridging
the sulfo-TAG-Drug and the Biotin-Drug bound to the streptavidin
surface will result in an ECL signal. After the final incubation,
the plate is washed with 0.05% Tween in PBS. Read buffer T 2.times.
is then added and the plate is read on a Sector PR2400. The
electro-chemiluminescent signal is proportional to the anti-drug
antibody in each sample. Sample results are converted to a signal
to background ratio (S/B) by dividing the average ECL signal from
an individual sample by the average ECL signal of the negative
control.
1.4 Bridging Immunoassay with Acid Dissociation Procedure
Samples are initially diluted 1:10 in assay buffer (300 mM Acetic
acid, 2% BSA) in incubated at 22-26.degree. C. for 45 minutes.
Samples are then added to a polypropylene plate in duplicate wells
and a solution containing equi-molar concentrations of Biotin-Drug
and sulfo-TAG-Drug in assay buffer in addition to Tris HCL is added
at a ratio to effectively neutralize the pH to 7.0. The plate is
then incubated for 2 hours in a 22-26.degree. C. shaker at 450 rpm.
A streptavidin coated MSD plate is blocked with assay buffer for a
minimum of one hour. Following the incubation, the MSD plate is
washed 3 times and 50 .mu.L of the incubated mixture is transferred
from the polypropylene plate to the MSD plate and incubated for 2
hours on a 22-26.degree. C. shaker. Following the incubation, the
plate is washed and a solution of MSD read buffer T containing
tripropylamine is added and the plate is read on the MSD sector
imager 2400.
Within the instrument, a voltage is applied. In the presence of
tripropylamine, sulfo-TAG.RTM. participates in an
electro-chemiluminescent (ECL) reaction. The antibodies bridging
the sulfo-TAG-Drug and the Biotin-Drug bound to the streptavidin
surface will result in an ECL signal. After the final incubation,
the plate is washed with 0.05% Tween in PBS. Read buffer T 2.times.
is then added and the plate is read on a Sector PR2400. The
electro-chemiluminescent signal is proportional to the anti-drug
antibody in each sample. Sample results are converted to a signal
to background ratio (S/B) by dividing the average ECL signal from
an individual sample by the average ECL signal of the negative
control.
1.5 PEG and Acid (PandA) Procedure 1
Samples are initially diluted 1/5 in assay buffer (300 mM Acetic
acid, 2% BSA) containing excess drug (10-50 .mu.g/mL) and incubated
for one hour at 37.degree. C. with 450 rpm in a polypropylene plate
to allow complexes between drug and any remaining free antibody in
the sample. This is followed by the addition of 3% PEG in Borate pH
8.0 to each sample and an overnight incubation at 2-8.degree. C.
The final concentration of PEG buffer in each sample is 1.5%.
The following day, the plate is centrifuged at 4000 rpms for 20
minutes to precipitate the complexes into a pellet. The pellets are
then re-suspended with 1.5% PEG in Borate pH 8.0 and centrifuged a
second time at 4000 rpms for 20 minutes. The wash cycle is repeated
three times. Following the final centrifugation, each sample
suspended in 100 .mu.l of 300 mM acetic acid and further diluted
1/10 (20 .mu.l sample+180 .mu.l acetic acid) for a final sample
dilution of 1/50. Diluted samples are then coated by adding 25
.mu.l in duplicate to wells of an MSD high bind plate and incubated
for one hour at 24.degree. C. shaking at 450 rpm. Following the
incubation, the plate is washed with 1.times. plate wash buffer and
blocked with 3% milk in PBS for one hour at 24.degree. C. shaking;
after which, the plate is washed and 100 ng/ml of sulfo-TAG-Drug
was added to the samples and incubated for one hour at 24.degree.
C. shaking. After the final incubation, the plate is washed with
0.05% Tween in PBS. Read buffer T 2.times. is then added and the
plate is read on a Sector PR2400. The electrochemiluminescent
signal is proportional to the anti-drug antibody in each
sample.
In some embodiments, the incubation step following the initial acid
addition can be carried out at 23.degree. C., 25.degree. C.,
27.degree. C., 30.degree. C., 32.degree. C., 35.degree. C.,
37.degree. C., 39.degree. C. or higher.
In some embodiments, following the final precipitation step, each
sample can be further diluted to a final sample dilution of, e.g.,
1:5, 1:10, 1:20, 1:25, 1:30, 1:40, 1:50, or 1:60, 1:80, 1:100, or
1:200. Typically the final sample dilution will be <1:100, which
is the minimal required dilution (MRD) set by the Food and Drug
Administration (FDA).
1.5 PEG and Acid (PandA) Procedure 2
Samples are initially diluted 1/5 in assay buffer (300 mM Acetic
acid, 2% BSA) containing excess drug (10-50 .mu.g/mL) and incubated
for one hour at 24.degree. C. with 450 rpm in a polypropylene plate
to allow complexes to form between drug and any remaining free
antibody in the sample. This is followed by the addition of a PEG
solution at a 1:1 PEG:sample ratio to each sample and an overnight
incubation at 2-8.degree. C. The final concentration of PEG was
optimized for each product to be optimal for precipitation of
specific complexes and achieving the desired specificity and
sensitivity while minimizing the effect of un-complexed molecules
such as non-specific IgGs. The PEG concentrations added to the
samples were between 3% and 6% (3% for drug A and 6% for drug B and
C added at a 1:1 ratio to diluted samples).
The following day, the plate is centrifuged at 4000 rpms for 30
minutes to precipitate the complexes into a pellet. The pellets are
then re-suspended with PEG in PBS and centrifuged a second time at
4000 rpms for 20 minutes. The wash cycle is repeated one additional
time. Following the final centrifugation, each sample is
re-suspended and diluted in 300 mM acetic acid to achieve the final
MRD desired for the particular assay ( 1/50 for Drug A and C and
1/25 for drug B). Samples are then coated in duplicate wells of an
MSD high bind plate. The plate is then incubated for one hour at
24.degree. C. shaking at 450 rpm. Following the incubation, the
plate is washed with 1.times. plate wash buffer and blocked with 3%
milk in PBS for one hour at 24.degree. C. shaking. The plate is
then washed and a solution containing sulfo-TAG-Drug is added to
the samples and incubated for one hour at 24.degree. C. shaking.
After the final incubation, the plate is washed with 1.times. plate
wash buffer. Read buffer T 2.times. is then added and the plate is
read on a Sector PR2400. The electro-chemiluminescent signal is
proportional to the anti-drug antibody in each sample.
In some embodiments, the incubation step following the initial acid
addition can be carried out at 23.degree. C., 25.degree. C.,
27.degree. C., 30.degree. C., 32.degree. C., 35.degree. C.,
37.degree. C., 39.degree. C. or higher.
In some embodiments, following the final precipitation step, each
sample can be further diluted to a final sample dilution of, e.g.,
1:5, 1:10, 1:20, 1:25, 1:30, 1:40, 1:50, or 1:60, 1:80, 1:100, or
1:200. Typically the final sample dilution will be <1:100, which
is the minimal required dilution (MRD) set by the Food and Drug
Administration (FDA).
2.1 Drug A
Sensitivity and Drug Tolerance Assessment
For Drug A, the PandA method was compared to the traditional MSD
bridging assay with and without acid dissociation to improve drug
tolerance. Assay sensitivity and drug Tolerance were determined
using affinity purified rabbit anti-drug at concentrations ranging
from 8 .mu.g/mL to 125 ng/mL with and without drug (Drug A) at
various concentrations (0, 0.1, 10 and 100 .mu.g/mL) in the MSD
bridging assay format with (FIG. 2A-B) and without acid (FIG. 3A-B)
dissociation. The samples containing ADA and drug were prepared in
pooled normal human sera and incubated for at least one hour at
37.degree. C. allowing drug/ADA complexes to form prior to
assaying.
Cut point was determined by evaluating 40 normal human serum
samples, calculating the mean and the standard deviation for the
samples and calculating the 95.sup.th percentile factor of
1.645-times the standard deviation and adding it to the mean as is
typically recommended. For drug A, the new method was compared to
the traditional bridging assay with and without acid dissociation
for drug tolerance.
FIG. 2 is a summary of the data comparing the MSD bridging assay
format without acid dissociation. The results indicate a strong
dose response for ADA detection in the absence of drug and
inhibition seen with 1 .mu.g/mL of drug. (FIG. 2A) FIG. 2B
indicates low recoveries of antibody detection, approximately 10%
at the 125 ng/mL of ADA at the lowest concentration of drug tested
of 1 .mu.g/mL. (FIG. 2B) The assay sensitivity was reduced from 15
ng/mL in the absence of drug to 342 ng/mL with 1 .mu.g/mL of drug.
The sensitivity in the presence of 100 .mu.g/mL of drug was reduced
to 5143 ng/mL or 5.1 .mu.g/mL.
FIG. 3 is a summary of the data from the MSD bridging assay format
with acid dissociation. The results indicate a similar dose
response to the bridging assay without acid for ADA detection in
the absence of drug and inhibition seen with 1 .mu.g/mL of drug.
(FIG. 3A) The percent recoveries remained acceptable with 1
.mu.g/mL of drug but reduced to 35% at the 125 ng/mL of ADA at the
10 .mu.g/mL of drug which is lower than the drug Cmax. (FIG. 3B)
The assay detection sensitivity was maintained for the 1 and 10
.mu.g/mL of drug at around 15 ng/mL while found to be 262 ng/mL in
the presence of 100 .mu.g/mL of drug. Although the sensitivity of
the assay meets some proposed guidelines of 250-500 ng/mL in this
method, this finding is specific to this product and antibody
combination and may not be acceptable for other products or with
different antibody controls.
FIGS. 4A and 4B are a summary of the data from the PandA (Procedure
2) precipitation format. The results indicate an acceptable dose
response in the absence of drug and no significant inhibition seen
due to drug present in the samples. (FIG. 4A) The percent
recoveries remained acceptable mostly between 80-120% regardless of
the drug amount present in the samples when compared to the sample
results with no drug as a reference. (FIG. 4B) Similarly, the assay
detection sensitivity was maintained at 9-14 ng/mL despite drug
present at 100 .mu.g/mL which is 3-4 folds higher than the expected
Cmax of Drug A.
Table 1 is a summary of the assay sensitivities for ADA detection
at various concentrations of Drug A tested in each of the methods
tested. The assay sensitivity concentrations were obtained by back
fitting the S/B cut point from each antibody curve shown in FIGS.
2A, 3A and 4A. ADA: Anti-drug antibody; S/B:
Signal-to-background.
TABLE-US-00001 TABLE 1 Assay Sensitivity ng/mL Bridging Assay
Bridging Assay Drug without Acid with Acid PandA (.mu.g/mL)
Dissociation Dissociation method 0 15 15 10 1 342 8 13 10 393 16 9
100 5143 262 14
As shown in Table 1, the PandA method not only improved the drug
tolerance but also maintained the assay sensitivity at 9-14 ng/mL
despite presence of 100 .mu.g/mL of Drug. Antibody detection
recovery remained mostly between 80 and 120% regardless of the
amount of drug present in the sample, which is superior to the
bridging immunoassay with acid dissociation. Titer Results
Comparison
The PandA method can also be used to accurately report antibody
titers in the presence of drug. To determine whether end point
titers correlate between samples that contain antibody alone (0
.mu.g/ml drug) and others that contain an equivalent amount of
antibody but high drug concentration (100 .mu.g/mL drug), test
samples were analyzed in the method where dilutions for titration
were performed following two different titration schemes. The first
scheme incorporated a titration prior to PEG precipitation and the
second scheme incorporated the titration prior to coating on the
high bind plates. The end point titers were identical between the
no drug and drug containing samples at the same antibody level as
well as between the titration schemes based on the assay titer cut
point value (S/B of 1.2). The end point titer data is summarized in
Table 2.
TABLE-US-00002 TABLE 2 Sample A B DRUG Level .mu.g/mL 100 0 100 0
Titer (diluted pre-PEG) 1600 1600 400 400 Titer (diluted post-PEG)
1600 1600 200 400
3.1. MSD High Bind Plates (Lot to Lot) and Overall Precision
To determine whether MSD High Bind plates contribute to assay
variability, coating of samples was performed on three different
lots. Data was plotted and analyzed for equivalence using
ANOVA.
To determine whether MSD High Bind plates contribute to the assay
variability, coating of samples was performed on three different
MSD plate lots. The samples contained affinity purified rabbit
antibody with various concentrations of drug. The observed S/B was
plotted against the ADA concentration for each of the three lots of
plates as shown in FIG. 5. The precision between plate lots was
found to be acceptable with CV less than 20% (Table 3) and ANOVA
showed no significant differences between the three lots with a
p-value of 0.1776.
4.1 Drug B
Target Interference in MSD Bridging Assay with Acid
Dissociation
For Drug B, a specific challenge was seen in the MSD bridging assay
with acid dissociation since the target for Drug B changes from a
monomer to a dimer at low pH causing false positive results. The
dimerization effect is seen in 100% of normal serum samples and
disease baseline samples in the MSD bridging assay with acid
dissociation. This phenomenon was similar to what was reported by
Dai et al. [Dai S, Schantz A, Clements-Egan A, Cannon M, Shankar G:
Development of a method that eliminates false-positive results due
to nerve growth factor interference in the assessment of fulranumab
immunogenicity. AAPS J. 16(3), 464-477 (2014)] where it was found
that high apparent incidence of anti-drug antibody (ADA) in phase 1
studies was the result of detection of drug target, a homodimer,
due to its ability to bridge drug molecules. Dai et al. found that
the acid-dissociation-based pretreatment of samples used for
mitigating drug interference dramatically increased drug target
interference.
To demonstrate the effect of endogenous target dimerization of the
drug target due to acid dissociation and false positive results in
the assay, normal (n=32) and baseline disease (n=16) serum samples
were analyzed in the bridging assay with and without acid
dissociation to highlight the effect of endogenous target
dimerization due to acid dissociation and false positive results in
the assay.
FIG. 6 is a representation of the normal serum samples tested in
the MSD bridging assay with and without acid dissociation. A normal
distribution at or near the background of the assay is observed
(population on the left) in the non-acid treated set while some
positive results were observed with S/B greater than 20 when acid
dissociation was applied to the bridging assay.
FIG. 7 is a representation of disease baseline serum samples when
analyzed in the MSD bridging assay without and with acid
dissociation as well as the PEG and Acid (PandA) method. Results
were comparable between the MSD bridging without acid treatment and
PandA method while the acid treatment resulted in higher S/B levels
for the majority of the samples tested suggesting interference from
drug target due to the dimerization effect at low pH.
As shown in FIGS. 6 and 7, for Drug B, the bridging assay with acid
dissociation is not a feasible approach in normal or disease
population due to the dimerization of the drug target at lower pH
causing false positive results for all samples with results
proportional to the amount of endogenous target. For that reason,
the assay sensitivity and drug tolerance for Drug B was only
compared between the PandA method and the existing MSD bridging
assay without acid dissociation.
FIGS. 8A-B and 9A-B are summaries of the data for Drug B comparing
the bridging assay format without acid dissociation to the PEG and
Acid method (PandA).
In FIG. 8A, the MSD bridging assay format resulted in an acceptable
dose response for ADA detection in the absence of drug and complete
inhibition seen with 10 .mu.g/mL and a decrease of sensitivity from
47 ng/mL in the absence of drug to 2 .mu.g/mL of antibody with 10
.mu.g/mL of drug.
For Drug B, the bridging assay without acid dissociation
sensitivity was validated at 50 ng/mL with poor drug tolerance.
Levels of drug at 250 ng/mL inhibited detection of anti-Drug B
antibodies at 250 ng/mL and levels of drug at 1 .mu.g/mL inhibited
detection of the antibody at 500 ng/mL.
FIGS. 9A-B are a summary of the data from the PandA precipitation
format. The results indicate an acceptable dose response in the
absence of drug and no inhibition seen due to drug present in the
samples. The percent recoveries remained acceptable mostly between
80-120% regardless of the drug amount present in the samples when
compared to the sample results with no drug as a reference. The
assay detection sensitivity was maintained at 39 to 63 ng/mL
despite drug present at 250 .mu.g/mL, which is higher than the
expected Cmax.
5.1 Drug C
Sensitivity and Drug Tolerance Assessment
For Drug C, the assay sensitivity and drug tolerance was compared
between the new method and the existing MSD bridging assay with
acid dissociation where expected drug tolerance cannot be
achieved.
FIGS. 10A-B and 11A-B are summaries of the data for Drug C
comparing the bridging assay format with acid dissociation to the
PEG and Acid method (PandA).
In FIGS. 10A-B, the MSD bridging assay format resulted in an
acceptable sensitivity in the absence of drug but inhibition was
seen with as little as 2.5 .mu.g/mL of drug despite acid treatment
in the bridging immunoassay. The sensitivity of the assay changed
from 227 ng/mL to 2788 ng/mL in the presence of 25 .mu.g/mL and
antibodies were completely undetectable in the presence of 250
.mu.g/mL of Drug, levels expected in some clinical samples.
FIGS. 11A-B shows the PandA results to be superior to the bridging
immunoassay with acid dissociation where antibody detection was
maintained with complete recovery even in the presence of 250
.mu.g/mL of drug. Percent recoveries were observed mostly between
80-120% regardless of the drug amount present in the samples when
compared to the sample results with no drug as a reference. The
assay detection sensitivity was maintained between 129-175 ng/mL
despite drug present at 250 .mu.g/mL, which is higher than what is
expected in clinical samples.
The examples above describe case studies for three humanized
monoclonal antibodies A-C (an IgG1 and 2 IgG4 drugs).
The three drug specific PandA ADA assays resulted in complete
recovery of ADA in samples containing drug levels in excess of
those expected in patients, in contrast to the commonly used assay
dissociation approach in MSD bridging assays. This breakthrough
novel method shows significant improvement over the current
approaches. In fact, the drug interference or under detecting of
ADA in all three cases were completely eliminated and this assay
principle could be used not only for ADA assays but also PK and
biomarker (drug target) analysis in the presence of interference
factors.
The PandA ADA assay method described herein was shown to be
effective at improving both detection and recovery of ADA in
samples containing interferent levels in excess of what is
clinically relevant. The method also reported consistent antibody
titers regardless of the amount of drug present. The method was
shown to be superior to the traditional solution based bridging
assay with acid dissociation maintaining the ADA detection
sensitivity at drug levels in excess of expected clinical Cmax
levels and no significant inhibition.
The PandA method can also be applied to PK assays where an ADA or
Drug Target is an interferent. Simply, excess antibody
(anti-Idiotype) or Target would be added to form complexes and
detection will be using a labeled anti-Idiotype that is specific to
the drug. In addition, the method can be applicable in drug target
biomarker assays with potential drug interference. In summary, the
inventors have described a novel application for PEG precipitation
of complexes that resolves drug and possibly target interferences
in an ADA immunoassay.
Many different methods and platforms have been used with limited
success to address circulating drug interference in immunoassays
for the detection of ADA. This disclosure a novel method that
employs PEG and Acid (PandA) that eliminates drug and possibly
target interferences in an ADA immunoassay. The novel method showed
complete elimination of drug interference at high drug
concentrations and demonstrated while maintaining assay sensitivity
in contrast to the traditional solution based MSD bridging assay
without or even with acid dissociation. By applying the following
assay components, the PandA assay has demonstrated its intended use
to sensitively and specifically detect ADA in the presence of drug
and/or drug target: addition of excess drug material to form
drug/ADA complexes; precipitation using polyethylene glycol to get
total ADA; acid dissociation and coating of reconstituted
precipitate in an acidic solution on a high bind carbon plate with
a large capacity to allow for binding to dissociated ADA; and
specific detection of the total ADA levels sulfo-TAG.RTM.
conjugated drug with an ECL output.
In addition, this method also reported consistent antibody titers
regardless of the amount of drug present in the sample showing
assay precision. Also reported, the method effectively resolve
target interference causing false positive results due to target
dimerization in MSD bridging immunoassays with acid
dissociation.
In summary, the method is superior to the traditional bridging with
acid dissociation method as evidenced by the three proof of
principle studies reported above where complete recovery and
detection of ADA in samples was achieved with high drug amounts in
the samples. This method was successfully validated according to
current regulatory expectations and clinical samples tested.
The PandA method described here has shown significant improvement
for ADA detection in the presence of excess drug. It has a broad
applications based on the principles: (1) saturate free analyte to
form all bound analyte in a complex and (2) precipitate the
complexes (3) acid dissociate to free analyte without
neutralization to reduce analyte re-bound while coating the free
analyte under acidic condition onto a large coating surface to
immobilize free analyte, and (4) detect free analyte using specific
reagent. In the examples above, the inventors have provided three
immunogenicity case studies to demonstrate the utility of this
novel technology.
Other Embodiments
It is to be understood that while the invention has been described
in conjunction with the detailed description thereof, the foregoing
description is intended to illustrate and not limit the scope of
the invention, which is defined by the scope of the appended
claims. Other aspects, advantages, and modifications are within the
scope of the following claims.
* * * * *
References